Naslov (srp)

Biohemijska karakterizacija bakterija izolovanih iz naftom zagađenih lokaliteta i njihova primena za bioremedijaciju i proizvodnju egzopolisaharida : doktorska disertacija

Autor

Žerađanin, Aleksandra, 1990-

Doprinosi

Jovančićević, Branimir, 1962-
Vujisić, Ljubodrag, 1978-
Beškoski, Vladimir, 1975-
Gojgić-Cvijović, Gordana, 1960-
Avdalović, Jelena, 1974-

Opis (srp)

Nafta i rafinisani proizvodi prerade nafte prilikom eksploatacije, transporta, skladištenja iakcidentalnih izlivanja zagađuju životnu sredinu i ispoljavaju toksične efekte po živi svet u njojuključujući i čoveka. S druge strane, naftom zagađena životna sredina predstavlja lokalitet u kome živebrojni mikroorganizmi koji imaju sposobnost degradacije različitih zagađujućih supstanci. Cilj ovedisertacije je bio ispitivanje primena različitih mikroorganizama izolovanih iz lokaliteta zagađenihnaftom, za dva procesa, bioremedijaciju i proizvodnju egzopolisaharida.Istraživanja vezana za bioremedijaciju su podeljena u dva dela. Prvi deo studije ispitujesposobnost izolovanog konzorcijuma mikroorganizama, degradera ugljovodonika, u procesimabioremedijacije izuzetno kompleksnih lokaliteta, kao što su isplačne jame. Isplačne jame predstavljajuzemljane rezervoare u koje se odlaže otpad, nastao tokom ekspoloatacije nafte i zemnog gasa. Napočetku procesa napravljen je bioremedijacioni supstrat dobijen mešanjem zemljišta sa ruba isplačnejame i rastresitog sedimenta bogatog naftnim ugljovodonicima. U bioremedijacioni supstrat je dodatizolovan konzorcijum mikroorganizama. Pokazano je da nakon 180 dana konzorcijum sa visokomefikasnošću razgrađuje naftne ugljovodonike i smanjuje njihovu koncentraciju za 95,3%. Ovako visokprocenat razgradnje ukazuje da je u konzorcijumu koji je korišćen u postupku bioremedijacije prisutnavelika količina raznovrsnih mikroorganizama što je metagenomskom analizom i potvrđeno. Naime,analiza je pokazala da su dominantno prisutni rodovi Gordonia, Bacteroides, CandidatusMacondimonas, Brucella, Parvibaculum, Mycobacterium. Navedeni rodovi su u literaturi ranijeistaknuti kao degraderi naftnih ugljovodonika. Prednost metagenomske analize se ogleda u tome, štopored mikroorganizama koji se mogu gajiti u laboratoriji, uspešno identifikuje i nekulturabilneautohtone mikroorganizme prisutne u uzorku.U drugom delu istraživanja proučavana je efikasnost ex situ postupka bioremedijacijekontaminiranog zemljišta, pored praćenja stepena mikrobiološke razgradnje naftnog polutanta, određenje i njegov grupni sastav, kao i promene u količini frakcija tokom studije. Dodatni kvalitet ovogistraživanja je i ispitivanje nastanka huminskih kiselina tokom procesa bioremedijacije. Tokompostupka, količina naftnih ugljovodonika je smanjena u visokom procentu, za 86,6% u odnosu napočetnu vrednost. Analizom uzoraka tokom bioremedijacije je utvrđeno da se sa procesombiodegradacije paralelno odvija i humifikacija. Pokazano je da je nakon 150 dana postupkabioremedijacije količina huminskih kiselina porasla za 26,5%, pri čemu su nastale huminske kiselinebile bogatije aromatičnim strukturama i kiseoničnim funkcionalnim grupama. Ovako obogaćenozemljište, ranije zagađen ekosistem koji je tretiran bioremedijacijom, ponovo omogućava životmikroorganizmima i biljkama, jer huminske kiseline u njemu pozitivno utiču na mikrobni rast i njihovmetabolizam, indukuju sintezu enzima i predstavljaju redoks aktivna jedinjenja.U disertaciji je izolovan i ispitivan mikroorganizam prisutan u sredini zagađenoj naftnimugljovodonicima, koji ima sposobnost produkcije egzopolisaharida. Mikroorganizam CH-KOV3,izolovan iz kanala otpadnih voda Pančevo, u preliminarnim laboratorijskim testovima je pokazaopotencijal za sintezu egzopolisaharida. Izolovana bakterija je u okviru teze molekularno identifikovanakao vrsta roda Brachybacterium, a sekvenca 16s rRNK zavedena u NCBI bazu podataka podpristupnim brojem KC881303.1. Daljom analizom izolata je utvrđeno da pripada grupi nepokretnihbakterija koje ne proizvode spore, a rastu u prisustvu kiseonika. Ovoj Gram-pozitivnoj bakteriji jepomoću 2D GCxGC-MS tehnike određen masnokiselinski profil ukupnih lipida, od kojih sudominantno prisutni bili ai-C15, i-C15, ai-C17, i-C17, i-C16, n-C16, n-C18 i C18:2 metil estri masnihkiselina. Izolat je okarakterisan i pomoću MALDI-TOF MS tehnike, a rezultat je značajan jer dobijenproteinski profil predstavlja jedinstven ˮotisak prstaˮ i do danas je jedini publikovan spektar rodaBrachybacterium.Tokom studija produkcije polisaharida upotrebom izolovane bakterije Brachybacterium sp. CH-KOV3 je utvrđeno da ona proizvodi najveću količinu egzopolisaharida pri temperaturi od 28 °C i pHvrednosti 7, na podlozi sa 500 g/L saharoze. Pri ovim uslovima je dobijeno 45,2 g/L egzopolisaharida,što je čak 5 puta veća količina u poređenju sa ranije publikovanim rezultatima za rod Brachybacterium.Detaljna strukturna analiza polisaharida pokazala je da je proizvedeni egzopolisaharid fruktanskog tipa,relativno visoke molekulske mase sa β(2,6) glikozidnom vezom u osnovnom nizu i grananjem upoložaju β(2,1), što odgovara egzopolisaharidu levanu

Opis (srp)

Hemija - Biohemija / Chemistry - Biochemistry Datum odbrane: 20. 07. 2022.

Opis (eng)

During their exploitation, transport, storage and accidental spills, oil and refined oil productspollute the environment and have toxic effects on living organisms including humans. On the otherhand, such oil-polluted environments are localities inhabited by numerous microorganisms that havethe ability to degrade some of the pollutants. The aim of this dissertation was to investigate theapplication of various microorganisms isolated from oil-contaminated sites for two processes,bioremediation and exopolysaccharide production.Research related to bioremediation included two studies. The first study examined theinvolvement of an isolated consortium of microorganisms, hydrocarbon degraders, in thebioremediation processes in extremely complex localities, such as mud pits. Mud pits are earthenreservoirs in which waste generated during the exploitation of oil and natural gas is disposed of. In thestudy, at the beginning of the process, a bioremediation substrate was obtained by mixing the soil fromthe edge of a mud pit with loose sediment rich in petroleum hydrocarbons. A consortium ofmicroorganisms was isolated, cultivated and then added to the bioremediation substrate. After 180 daysof bioremediation, the consortium decomposed petroleum hydrocarbons with high efficiency andreduced their concentration by 95.3%. Such a high percentage of hydrocarbon degradation indicatesthat large numbers of various microorganisms are present in the consortium used in the bioremediationprocess, which was confirmed by metagenomic analysis. Namely, the analysis showed that the generaGordonia, Bacteroides, Candidatus Macondimonas, Brucella, Parvibaculum and Mycobacteriumpredominate in the consortium. These genera have previously been highlighted in the literature aspetroleum hydrocarbon degraders. The advantage of metagenomic analysis is reflected in the fact thatin addition to microorganisms that can be grown in the laboratory, it successfully identifies unculturedindigenous microorganisms present in the matrix.In the second study, on the efficiency of ex situ bioremediation of contaminated soil, in additionto monitoring the degree of microbiological degradation of petroleum pollutants, the groupcomposition of the petroleum pollutants was determined, as well as changes in the amounts of fractionsduring the study. An additional quality of this research is the investigation of the formation of humicacids during the bioremediation process. During the bioremediation procedure, the amount ofpetroleum hydrocarbons in the soil was reduced by a high percentage, i.e. by 86.6% compared to theinitial value. Analysis of soil samples taken during bioremediation showed that humification takesplace in parallel with the biodegradation process. In fact, after 150 days of the bioremediation process,the amount of humic acids increased by 26.5%, with the resulting humic acids being richer in aromaticstructures and oxygen functional groups than at the start. This resulting enriched soil, obtained from apreviously polluted ecosystem that was treated by bioremediation, allows microorganisms and plants tothrive, because it contains humic acids that have a positive effect on microbial growth and metabolism,induce enzyme synthesis and act as redox active compounds.A microorganism from a petroleum hydrocarbon-contaminated environment which couldproduce exopolysaccharides was isolated and examined. The microorganism CH-KOV3, isolated fromthe Pančevo wastewater canal, showed potential to synthesize exopolysaccharides in preliminarylaboratory tests. The isolated bacterium was molecularly identified as a species belonging to genusBrachybacterium, and the 16s rRNA sequence was entered into the NCBI database under accessionnumber KC881303.1. Further analysis of the isolate determined that it belongs to the group ofimmobile bacteria that do not produce spores, but grow in the presence of oxygen. This Gram-positivebacterium was studied by the 2D GCxGC-MS technique to determine the fatty acid profile of its totallipids; predominant lipids are ai-C15, i-C15, ai-C17, i-C17, i-C16, n-C16, n-C18 and C18:2 fatty acidmethyl esters. The isolate was also characterized by the MALDI-TOF MS technique, and the result issignificant because the protein profile obtained represents a unique fingerprint and, to date, is the onlypublished spectrum for the genus Brachybacterium.Studies into polysaccharide production using the isolated Brachybacterium sp. CH-KOV3showed it produces the largest amount of exopolysaccharides at 28 °C and pH 7, on a medium with 500g/L sucrose. Under these conditions, 45.2 g/L of exopolysaccharide were obtained, which is five timeshigher than the previously published results for genus Brachybacterium. Detailed structural analysisshowed the exopolysaccharide produced by Brachybacterium CH-KOV3 was a fructan-typepolysaccharide of relatively high molecular weight, with β(2,6) glycosidic bonds in the main chain andbranching in the β(2,1) position, which corresponds to the exopolysaccharide levan

Jezik

srpski

Datum

2022

Licenca

Ovo delo je licencirano pod uslovima licence
Creative Commons CC BY-NC-ND 3.0 AT - Creative Commons Autorstvo - Nekomercijalno - Bez prerada 3.0 Austria License.

http://creativecommons.org/licenses/by-nc-nd/3.0/at/legalcode

Predmet

OSNO - Opšta sistematizacija naučnih oblasti, Organska hemija

OSNO - Opšta sistematizacija naučnih oblasti, Biohemija

bioremedijacija; ugljovodonici; humifikacija; Brachybacterium; levan

OSNO - Opšta sistematizacija naučnih oblasti, Organska hemija

OSNO - Opšta sistematizacija naučnih oblasti, Biohemija

bioremedijacija; ugljovodonici; humifikacija; Brachybacterium; levan.